Enzymatic Synthesis of Region-Specific Isotope-Labeled DNA Oligomers for NMR Analysis

The recent development of an enzymatic procedure for large scale synthesis of DNA oligomers uniformly labeled with the stable isotopes super(13)C and super(15)N will undoubtedly advance the application of NMR spectroscopy to structural studies of DNA in the same way that uniform labeling by in vitro transcription has led to the recent quantum leap in NMR structural characterization of RNA. As first demonstrated for proteins, uniform super(13)C and super(15)N enrichment significantly improves spectral resolution by allowing the dispersion or filtering of super(1)H frequencies via scalar coupling with the heteronuclei. However, most of this benefit from heteronuclear labeling is lost for nucleic acids larger than similar to 30-40 residues because the limited library of nucleic acid residues results in extensive spectral overlap for all three nuclei. Additional limitations arise due to the increased relaxation rates of larger oligonucleotides, which is exacerbated by the heteronuclear dipolar couplings. These problems can be circumvented by regional labeling of the oligonucleotide, i.e., labeling of only a subset of residues within one oligonucleotide. In this communication we present a general method for region-specific labeling of DNA and its application to the study of a 64-residue DNA crossover structure that serves as a model for the Holliday junction intermediate in genetic recombination and repair. Although the global structural features of such branched structures have been studied by a variety of techniques, little is known about the details of DNA conformation at junctions. With specific labeling of the crossover region, this structural information can be obtained from NMR.